Pulmonary fibrosis (PF) is a chronic and refractory interstitial lung disease with limited therapeutic options. 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate, has been shown to have anti-oxidative and anti-inflammatory properties. However, the effect and the underlying mechanism of 4-OI on PF are still unknown. WT or Nrf2 knockout (Nrf2-/-) mice were intratracheally injected with bleomycin (BLM) to establish PF model and then treated with 4-OI. The mechanism study was performed by using RAW264.7 cells, primary macrophages, and conditional medium-cultured MLE-12 cells. 4-OI significantly alleviated BLM-induced PF and EMT process. Mechanism studies have found that 4-OI can not only directly inhibit EMT process, but also can reduce the production of TGF-β1 by restraining macrophage M2 polarization, which in turn inhibits EMT process. Moreover, the effect of 4-OI on PF and EMT depends on Nrf2. 4-OI ameliorates BLM-induced PF in an Nrf2-dependent manner, and its role in alleviating PF is partly due to the direct inhibition on EMT, and partly through indirect inhibition of M2-mediated EMT. These findings suggested that 4-OI has great clinical potential to develop as a new anti-fibrotic agent for PF therapy.

BackgroundEndotoxin tolerance (ET) is a protective mechanism in the process of sepsis, septic shock, and their sequelae including uncontrolled inflammation. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what and how T-cell development contributes to ET inductions remain unclear.MethodsMice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of PBS was used as a control. We performed high-throughput sequencing to analyze the characteristics and changes of CD4+SP TCRβ CDR3 repertoires with respect to V direct to J rearrangement during the ET induction. Moreover, the proportion and proliferation, as well as surface molecules such as CD80 and CD86, of F4/80+ macrophages were analyzed using FCM. Furthermore, ACT assay was designed and administered by the tail vein into murine LPS-induced mouse model to evaluate the role of F4/80+ macrophages on the development of CD4+SP thymocytes in ET condition.ResultsWe found that the frequency and characteristics of the TCRβ chain CDR3 changed obviously under condition of ET, indicating the occurrence of TCR rearrangement and thymocyte diversification. Moreover, the absolute numbers of F4/80+ macrophages, but not other APCs, were increased in thymic medulla at 72-h group, accompanied by the elevated function-related molecules of F4/80+ macrophages. Furthermore, adoptively transferred OVA332-339 peptide-loaded macrophages into Rag-1−/− mice induced the clone deletion of OVA-specific CD4+SP, thereby ameliorating the pathology in lung tissue in LPS challenge.ConclusionsThese data reveal that the frequency and characteristics of the TCRβ chain CDR3 undergo dynamic programming under conditions of LPS tolerance. Furthermore, the peripheral macrophages may be a key factor which carry peripheral antigen to thymic medulla and affect the negative selection of T-cell population, thereby contributing to the formation of ET. These results suggest that the clone selection in thymus in ET may confer protection against microbial sepsis.

ObjectivesExcessive inflammatory responses and apoptosis are critical pathologies that contribute to sepsis-induced acute kidney injury (SI-AKI). Annexin A1 (ANXA1), a member of the calcium-dependent phospholipid-binding protein family, protects against SI-AKI through its anti-inflammatory and antiapoptotic effects, but the underlying mechanisms are still largely unknown.MethodsIn vivo, SI-AKI mouse models were established via caecal ligation and puncture (CLP) and were then treated with the Ac2-26 peptide of ANXA1 (ANXA1 (Ac2-26)), WRW4 (Fpr2 antagonist) or both. In vitro, HK-2 cells were induced by lipopolysaccharide (LPS) and then treated with ANXA1 (Ac2-26), Fpr2–siRNA or both.ResultsIn the present study, we found that the expression levels of ANXA1 were decreased, and the expression levels of TNF-α, IL-1β, IL-6, cleaved caspase-3, cleaved caspase-8 and Bax were significantly increased, accompanied by marked kidney tissue apoptosis in vivo. Moreover, we observed that ANXA1 (Ac2-26) significantly reduced the levels of TNF-α, IL-1β and IL-6 and cleaved caspase-3, cleaved caspase-8, FADD and Bax and inhibited apoptosis in kidney tissue and HK-2 cells, accompanied by pathological damage to kidney tissue. Seven-day survival, kidney function and cell viability were significantly improved in vivo and in vitro, respectively. Furthermore, the administration of ANXA1 (Ac2-26) inhibited the CLP- or LPS-induced phosphorylation of PI3K and AKT and downregulated the level of NF-κB in vivo and in vitro. Moreover, our data demonstrate that blocking the Fpr2 receptor by the administration of WRW4 or Fpr2–siRNA reversed the abovementioned regulatory role of ANXA1, accompanied by enhanced phosphorylation of PI3K and AKT and upregulation of the level of NF-κB in vivo and in vitro.ConclusionsTaken together, this study provides evidence that the protective effect of ANXA1 (Ac2-26) on SI-AKI largely depends on the negative regulation of inflammation and apoptosis via the Fpr2 receptor.

The present study was designed to explore the potential regulatory mechanism between mitophagy and pyroptosis during sepsis-associated acute lung injury (ALI). In vitro or in vivo models of sepsis-associated ALI were established by administering lipopolysaccharide (LPS) or performing caecal ligation and puncture (CLP) surgery. Pyroptosis levels were detected by electron microscopy, immunofluorescence, flow cytometry, western blotting and immunohistochemistry. Dual-luciferase reporter gene assay was applied to verify the targeting relationship between miR-138-5p and NLRP3. Methylation-specific PCR and chromatin immunoprecipitation assays were used to determine methylation of the miR-138-5p promoter. Mitophagy levels were examined by transmission electron microscopy and western blotting. NLRP3 inflammasome silencing alleviated alveolar macrophage (AM) pyroptosis and septic lung injury. In addition, we confirmed the direct targeting relationship between miR-138-5p and NLRP3. Overexpressed miR-138-5p alleviated AM pyroptosis and the pulmonary inflammatory response. Moreover, the decreased expression of miR-138-5p was confirmed to depend on promoter methylation, while inhibition of miR-138-5p promoter methylation attenuated AM pyroptosis and pulmonary inflammation. Here, we discovered that an increased cytoplasmic mtDNA content in sepsis-induced ALI models induced the methylation of the miR-138-5p promoter, thereby decreasing miR-138-5p expression, which may activate the NLRP3 inflammasome and trigger AM pyroptosis. Mitophagy, a form of selective autophagy that clears damaged mitochondria, reduced cytoplasmic mtDNA levels. Furthermore, enhanced mitophagy might suppress miR-138-5p promoter methylation and relieve the pulmonary inflammatory response, changes that were reversed by treatment with isolated mtDNA. In summary, our study indicated that mitophagy induced the demethylation of the miR-138-5p promoter, which may subsequently inhibit NLRP3 inflammasome, AM pyroptosis and inflammation in sepsis-induced lung injury. These findings may provide a promising therapeutic target for sepsis-associated ALI.

Myocardial infarction (MI) caused by ischemic cardiomyocyte necrosis induces inflammatory responses that strongly affect ventricular remodeling. Tolerogenic dendritic cells (tDCs) can suppress this effect on inflammatory responses. However, the precise role of atorvastatin-induced tDCs in ventricular remodeling after MI remains unclear. To explore the effect of necrotic cardiomyocytes (SNC) and/or atorvastatin on DC function, the expression of CD40, CD80, CD86, and MHC-II was determined using flow cytometry. The protein levels of TLR-4/NF-κB-related molecules were evaluated using western blotting. The infarct area after MI was determined via 2,3,5-triphenyltetrazolium chloride staining. The TUNEL assay was employed to evaluate the apoptosis of cardiomyocytes in heart sections. Masson's trichrome method was used to determine the extent of fibrosis. Compared to the DCs co-cultured with PBS (control), cells co-cultured with Supernatant-IM or Supernatant-NH produced higher levels of inflammatory cytokines, including TNF-α, IL-1, IL-6, IL-12P40, and IL-8. This cytokine production was impaired by atorvastatin treatment. SNC treatment induced DC maturation and enhanced inflammatory cytokine secretion and oxidative stress through TLR-4/NF-κB pathway activation. Compared to that in the PBS-treated group, the left ventricular ejection fraction was significantly improved after tDC treatment. Additionally, compared to that in the PBS-treated group, tDC treatment reduced the left ventricular end-diastolic and end-systolic diameters in mice. Furthermore, treatment with tDCs improved the left ventricular systolic function, attenuated inflammatory cell infiltration, and reduced cardiomyocyte apoptosis, myocardial fibrosis, and infarct size compared to those in the control group. Adoptive transfer of atorvastatin-induced tDCs alleviated post-infarction cardiomyocyte apoptosis and myocardial fibrosis in association with decreased inflammatory cell infiltration and inhibited oxidative stress, likely by suppressing TLR-4/NF-κB activation after myocardial infarction.

Cardiovascular diseases (CVD) have been associated with high exposure to fine particulate air pollutants (PM2.5). Alveolar macrophages are the first defense against inhaled particles. As soon as they phagocytize the particles, they reach an inflammatory phenotype, which affects the surrounding cells and associates with CVD. Not coincidentally, CVD are marked by a depleted heat shock response (HSR), defined by a deficit in inducing 70-kDa heat shock protein (HSP70) expression during stressful conditions. HSP70 is a powerful anti-inflammatory chaperone, whose reduced levels trigger a pro-inflammatory milieu, cellular senescence, and a senescence-associated secretory phenotype (SASP). However, whether macrophage senescence is the main mechanism by which PM2.5 propagates low-grade inflammation remains unclear. In this article, we review evidence supporting that chronic exposure to PM2.5 depletes HSR and determines the ability to solve the initial stress. When exposed to PM2.5, macrophages increase the production of reactive oxygen species, which activate nuclear factor-kappa B (NF-κB). NF-κB is naturally a pro-inflammatory factor that drives prostaglandin E2 (PGE2) synthesis and causes fever. PGE2 can be converted into prostaglandin A2, a powerful inducer of HSR. Therefore, when transiently activated, NF-κB can trigger the anti-inflammatory response through negative feedback, by inducing HSP70 expression. However, when chronically activated, NF-κB heads a set of pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, unfolded protein response, inflammasome activation, and apoptosis. During chronic exposure to PM2.5, cells cannot properly express sirtuin-1 or activate heat shock factor-1 (HSF-1), which delays the resolution phase of inflammation. Since alveolar macrophages are the first immune defense against PM2.5, we suppose that the pollutant impairs HSR and, consequently, induces cellular senescence. Accordingly, senescent macrophages change its secretory phenotype to a more inflammatory one, known as SASP. Finally, macrophages' SASP would propagate the systemic inflammation, leading to endothelial dysfunction and atherosclerosis.

Systemic inflammation plays an important role in cancer cachexia. However, among the systemic inflammatory biomarkers, it is unclear which has optimal prognostic value for cancer cachexia. The Kaplan-Meier method was used and the log-rank analysis was performed to estimate survival differences between groups. Cox proportional hazard regression analyses were conducted to assess independent risk factors for all-cause mortality. The C-reactive protein-to-albumin ratio (CAR) was the optimal prognostic assessment tool for patients with cancer cachexia, with 1-, 3-, and 5-year predictive powers of 0.650, 0.658, and 0.605, respectively. Patients with a high CAR had significantly lower survival rates than those with a low CAR. Moreover, CAR can differentiate the prognoses of patients with the same pathological stage. Cox proportional risk regression analyses showed that a high CAR was an independent risk factor for cancer cachexia. For every standard deviation increase in CAR, the risk of poor prognosis for patients with cancer cachexia was increased by 20% (hazard ratio = 1.200, 95% confidence interval = 1.132-1.273, P < 0.001). CAR is an effective representative of systemic inflammation and a powerful factor for predicting the life function and clinical outcome of patients with cancer cachexia.

In this study, we describe the kinetics of a new potential inflammatory biomarker, presepsin, together with a panel of well-established biomarkers in a human endotoxemia study. We evaluated biomarker correlations and identified combinations that could hold valuable insights regarding the state of infection.

BackgroundInflammation and metabolic syndrome (MetS) may act synergistically and possibly accelerate the initiation and progression of colorectal cancer (CRC). We prospectively examined the joint effect of MetS and inflammation on the risk of CRC.MethodsWe studied 92,770 individuals from the Kailuan study. MetS was defined based on the presence of three or more of the following components. (1) high glucose: FPG > 5.6 mmol/L; (2) high blood pressure: SBP ≥ 130 mmHg or DBP ≥ 85 mmHg; (3) high triglycerides: triglycerides > 1.69 mmol/L; (4) low HDL-C: HDL-C < 1.04 mmol/L in men or 1.29 mmol/L in women; and (5) visceral adiposity: waist circumference ≥ 85 cm in men or 80 cm in women. Inflammation was defined as hs-CRP ≥ 3 mg/L. We divided participants into four groups for the primary exposure according to the presence/absence of inflammation and presence/absence of MetS. Cox proportional hazards regression models were used to evaluate the association of MetS and/or inflammation with the risk of CRC.ResultsCompared with metabolically healthy noninflammatory individuals, inflammatory participants without MetS and inflammatory participants with MetS were associated with a 1.3-fold and 4.18-fold increased risk of CRC with corresponding HRs (95% CI) of 1.34 (1.09, 1.64) and 4.18 (3.11, 5.62), respectively. The combination of MetS and inflammation was associated with the highest risk of CRC in all subgroups, especially among participants who were female, in younger age, and obese. Sensitivity analyses further validated our primary findings.ConclusionsWe found the combination of MetS and inflammation could significantly increase the risk of CRC. Including CRP in the diagnosis of MetS may help to identify additional high-risk participants who should be targeted for early diagnosis and prevention of CRC.Trial registration Kailuan study, ChiCTR–TNRC–11001489. Registered 24 August, 2011-Retrospectively registered, http:// www.chictr.org.cn/showprojen.aspx?proj=8050

Exosomes derived from bone mesenchymal stem cells (BMSCs) are potential candidates for inflammatory bowel disease (IBD) treatment. The present study investigated the therapeutic effect and potential mechanism of BMSCs-derived exosomes on pyroptosis in IBD. We induced IBD in mice and cell models through dextran sulfate sodium (DSS) and LPS, respectively. The mRNA and protein expression levels were assessed by qRT-PCR, Western blotting, IF and IHC. The concentrations of IL-1β, IL-18 and TNFα were assessed using ELISA. ROS levels were determined using DCFH-DA staining. Cell proliferation of mIECs was analysed using an MTT assay. In addition, a flow cytometry assay was performed to detect pyroptosis. Finally, the binding relationship between miR-539-5p and NLRP3 was verified by a dual luciferase reporter gene assay. Our results revealed that intraperitoneal injection of BMSCs-derived exosomes inhibited DSS-induced pyroptosis as well as IBD symptoms in mice. In addition, BMSCs-derived exosome treatment suppressed pyroptosis, ROS levels and the concentrations of proinflammatory cytokines (IL-1β, IL-18 and TNFα) in LPS-treated mIECs in a miR-539-5p-dependent manner. Further research found that miR-539-5p suppressed NLRP3 expression in mIECs by directly targeting NLRP3. As expected, pyroptosis in LPS-treated mIECs was significantly reduced by NLRP3 knockdown. In addition, NLRP3 silencing restored the inhibitory effect of exosomes derived from BMSCs transfected with miR-539-5p inhibitor on pyroptosis in LPS-treated mIECs. The present study demonstrated that BMSCs-derived exosomal miR-539-5p suppresses pyroptosis through NLRP3/caspase-1 signalling to inhibit IBD progression.